This proposal describes single molecule kinetic and dynamic investigations of critical protein/nucleic acid intermediates in the reverse transcription mechanism of HIV-1. HIV-1 utilizes the nucleocapsid protein (NC), a nucleic acid chaperone, to facilitate a variety of steps critical to the reverse transcription of its RNA genome. In the first step of reverse transcription, HIV-1 uses tRNA(Lys,3) to prime minus strand DNA synthesis. NC has been shown to induce the unwinding of tRNA(Lys,3) and to facilitate its annealing to the RNA genome. NC again catalyzes nucleic acid rearrangements in two subsequent strand transfer steps critical to reverse transcription. The molecular-level picture of how NC executes its nucleic acid chaperone functions is unclear and will be elucidated by the fluorescence single molecule spectroscopy (SMS) methods in the proposed work, including fluorescence resonance energy transfer studies on various immobilized dyelabeled NC, RNA, and DNA model compounds and their complexes. In particular, SMS will be used to determine the kinetics and mechanisms of NC-induced unwinding and annealing of base-paired DNAs/RNAs to the HIV-1 RNA genome. The sequence of molecular rearrangements, the rate-limiting kinetic steps, and the molecularity of important reaction intermediates will be revealed using SMS methods. More generally, this work strives to develop powerful new strategies for molecular-level biochemical investigations of HIV-1 and related systems with highly heterogeneous kinetics.